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The Journal of Neuroscience, February 1, 2002, 22(3):1146–1154

Motivational Effects of Are Mediated by ␮- and ␬-Opioid Receptors

Sandy Ghozland,1 Hans W. D. Matthes,2 Frederic Simonin,2 Dominique Filliol,2 Brigitte L. Kieffer,2 and Rafael Maldonado1 1Laboratori de Neurofarmacologia, Facultat de Cie´ ncies de la Salut i de la Vida, Universitat Pompeu Fabra, 08003 Barcelona, Spain, and 2Centre National de la Recherche Scientifique Unite´ Propre de Recherche 9050, Ecole Superieure de Biotechnologie de Strasbourg, 67400 Illkirch, France

Repeated THC administration produces motivational and so- place conditioning protocols that reveal both THC rewarding matic adaptive changes leading to dependence in rodents. To and aversive properties. Absence of ␮ receptors abolishes THC investigate the molecular basis for dependence place preference. Deletion of ␬ receptors ablates THC place and its possible relationship with the endogenous opioid sys- aversion and furthermore unmasks THC place preference. tem, we explored ⌬9- (THC) activity in Thus, an opposing activity of ␮- and ␬-opioid receptors in mice lacking ␮-, ␦-or␬- genes. Acute THC- modulating reward pathways forms the basis for the dual eu- induced , antinociception, and hypolocomotion re- phoric–dysphoric activity of THC. mained unaffected in these mice, whereas THC tolerance and withdrawal were minimally modified in mutant animals. In con- Key words: ⌬9-tetrahydrocannabinol; place preference; place trast, profound phenotypic changes are observed in several aversion; knock-out; tolerance; dependence; reward

Cannabinoids and are the most widely consumed illicit to precipitate abstinence in -dependent rats (Navarro et worldwide (Smart and Ogborne, 2000). Both types of com- al., 1998). Besides, the severity of was reduced pounds mimic endogenous ligands and act through distinct by the administration of THC (Hine et al., 1975; Valverde et al., G-protein-coupled receptor families known as cannabinoid 2001) or the endogenous cannabinoid (Vela (Felder and Glass, 1998) and opioid (Kieffer, 1995) receptors. et al., 1995). This bidirectional cross-dependence has been re- Pharmacological studies have shown functional interactions be- cently confirmed by using knock-out mice, because opioid depen- tween the two systems (Manzanares et al., 1999). Thus, cannabi- dence was reduced in mice lacking the CB1 cannabinoid receptor noid and opioid share several pharmacological proper- (Ledent et al., 1999), whereas cannabinoid dependence was re- ties, including antinociception and hypothermia (Narimatsu et al., duced in mice lacking the preproenkephalin gene (Valverde et al., 1987; Vivian et al., 1998). Biochemical studies have revealed that 2000). repeated THC administration increases opioid gene ex- Another important aspect of marijuana activity is the complex- pression (Corchero et al., 1997a,b). Acute THC also increases ity of evoked emotional responses and in particular the possibility extracellular levels of endogenous in the nucleus of dual – effects (Halikas et al., 1985). Electri- accumbens (Valverde et al., 2001). The existence of cross- cal brain stimulation (Gardner et al., 1988) and in vivo microdi- tolerance between opioid and cannabinoid agonists has been alysis (Chen et al., 1990; Tanda et al., 1997) have suggested that supported by a variety of studies. Thus, morphine-tolerant ani- cannabinoids produce their rewarding action by stimulating me- mals show decreased THC antinociceptive responses, whereas solimbic transmission, a common substrate for the THC-tolerant rodents show a decrease in morphine antinocicep- rewarding effects of other substances of abuse (Koob, 1992), and tion (Hine, 1985; Thorat and Bhargava, 1994). Cross-dependence that ␮-opioid receptors could be involved (Tanda et al., 1997). between opioid and cannabinoid compounds has also been re- The endogenous cannabinoid system participates in the reward- ported. Indeed, the precipitated a ing effects of opioids, because both morphine self-administration withdrawal syndrome in THC-tolerant rats (Kaymakcalan et al., (Ledent et al., 1999) and place preference (Martin et al., 2000) 1977), whereas the cannabinoid antagonist SR171416A was able are decreased in mice lacking the CB1 receptor. However, the possible involvement of the endogenous opioid system in the different motivational responses induced by cannabinoids re- Received July 23, 2001; revised Nov. 7, 2001; accepted Nov. 15, 2001. mains to be clarified. GABAergic (Onaivi et al., 1990) and This work was supported by the European Commission (Biomed-2 Grant 98-2227, to R.M.), Dr. Esteve S. A. Laboratories (R.M.), Generalitat de Catalunya (Research corticotropin-releasing factor (Rodriguez de Fonseca et al., 1996) Distinction, to R.M.), the Spanish Ministry of Health (Fondo de Investigacio´n systems have been suggested to be involved in the anxiogenic Sanitaria Grant 99/0624, to R.M.), the Mission Interministerielle de Lutte contre la responses induced by cannabinoids. These anxiogenic effects Drogue et la Toxicomanie (B.K.), and the Centre National de la Recherche Scientifique (B.K.). We thank J. F. Poirier and N. Scallon for animal care. could have some influence in the dysphoric properties of canna- Correspondence should be addressed to Rafael Maldonado, Laboratori de binoids, but the mechanisms that underlie the potential aversive Neurofarmacologia, Facultat de Cie´ncies de la Salut i de la Vida, Universitat effects of THC remain unexplored. Pompeu Fabra, c/o Dr Aiguader 80, 08003 Barcelona, Spain. E-mail: [email protected]. To investigate these major aspects of cannabinoid–opioid in- Copyright © 2002 Society for Neuroscience 0270-6474/02/221146-09$15.00/0 teractions, we have examined whether the genetic ablation of Ghozland et al. • Cannabinoid Responses in Opioid Knock-Out Mice J. Neurosci., February 1, 2002, 22(3):1146–1154 1147

␮-opioid (Matthes et al., 1996), ␦-opioid (Filliol et al., 2000), or weight, as previously described (Koob et al., 1992): 0.9 point for the ␬-opioid (Simonin et al., 1998) receptors in mice has any influ- appearance of each checked sign in each 5 min time period; 0.4 point for each bout of counted sign. ence on THC tolerance, , and motivational Statistics. Acute effects and global withdrawal scores were compared by responses. using two-way ANOVA (genotype and treatment) between subjects followed by one-way ANOVA for individual differences. Values of tol- MATERIALS AND METHODS erance studies were compared by using three-way ANOVA (genotype and treatment as between groups factors and day as within group factor), Mice. The generation of mice lacking either ␮-opioid (MOR Ϫ/Ϫ), ␦ Ϫ Ϫ ␬ Ϫ Ϫ followed by corresponding two-way and one-way ANOVAs and post hoc -opioid (DOR / ), or -opioid (KOR / ) receptors has been comparisons when applied. described previously (Matthes et al., 1996; Simonin et al., 1998; Filliol et Place conditioning. An unbiased place conditioning procedure was used al., 2000). Mice weighing 22–24 gm at the start of the study were housed, to evaluate both rewarding and aversive properties of THC (Valjent and grouped, and acclimatized to the laboratory conditions (12 hr light/dark Ϯ Ϯ Maldonado, 2000) in animals lacking each opioid receptor. The appara- cycle, 21 1°C room temperature, 65 10% humidity) 1 week before tus consisted of two main square conditioning compartments (15 ϫ 15 ϫ the experiment with ad libitum access to food and water. All animals 15 cm) separated by a triangular central division (Maldonado et al., were 1:1 hybrids from 129/SV and C57B1/6 mouse strains. Wild-type 1997). The light intensity within the conditioning chambers was 50 Ϯ 5 littermates were used for the control groups in all experiments. Mutants lux. The movement and location of the mice were recorded by comput- and their wild-type littermates showed comparable spontaneous locomo- Ϫ Ϫ erized monitoring software (Videotrack; View Point, Lyon, France) with tor activity, except for DOR / mice, which displayed significant images relayed from a camera placed above the apparatus. During the hyperlocomotion (increase of 161.85 Ϯ 19.58% comparing with wild-type ϭ Ͻ preconditioning phase, -naive mice were placed in the middle of the controls, F(1,23) 6.277, p 0.05) as previously reported (Filliol et al., central division and had ad libitum access to both compartments (striped 2000). Behavioral tests and animal care were conducted in accordance and dotted compartment) of the conditioning apparatus for 20 min, with with the standard ethical guidelines (National Institutes of Health, 1995; the time spent in each compartment being recorded. The conditioning Council of Europe, 1996) and approved by the local ethical committee. phase consisted of five pairings with THC and five pairings with vehicle The observer was blind to the genotype and treatment in all experiments. for a 45 min conditioning time in all experiments. Mice were injected Drugs. THC (Sigma, Poole, UK) was dissolved in a solution of 5% with vehicle or THC (1 and 5 mg/kg, i.p.) and then immediately confined ethanol, 5% cremophor El, and 90% distilled water, and injected in a to the conditioning compartment. Treatments were counterbalanced as volume of 0.1 ml per 10 gm body weight. The selective CB1 cannabinoid closely as possible between compartments. Control animals received SR141716A was dissolved in a solution of 10% vehicle every day. The test phase was conducted exactly as the precon- ethanol, 10% cremophor El, and 80% distilled water, and injected by ditioning phase, i.e., ad libitum access to each compartment for 20 min. intraperitoneal route in a volume of 0.2 ml per 10 gm body weight. Mice conditioned with the dose of 1 mg/kg of THC received a single Tolerance and withdrawal. Animals were injected intraperitoneally THC (1 mg/kg, i.p.) injection in the home cage 24 hr before starting the twice daily at 9:00 A.M. and 7:00 P.M. for5dwithTHC(20mg/kg)or conditioning procedures, to avoid the dysphoric effects of the first drug vehicle. On day 6, mice only received the morning injection. Four exposure (Valjent and Maldonado, 2000). As previously described (Sti- different responses were measured once a day during the chronic THC nus et al., 1990; Valverde et al., 1996; Maldonado et al., 1997), the time treatment: body weight, rectal temperature, antinociception, and loco- in central area was proportionally shared and added to the time value of motor activity. Body weights were recorded for each animal, using an each conditioned compartment, as follows: the time spent in each com- electronic balance (Mettler PM 4800; sensitive to 0.01 gm), once a day partment is multiplied by the total time (1200) and divided by the total before morning injections. Locomotor measurements for each mouse time minus the time spent in the central area: were taken 20 min after morning injections by placing animals in indi- ϫ ϫ vidual actimeters (9 20 11 cm) (Imetronic, Bordeaux, France) Time in conditioned compartment ϫ equipped with two lines of six infrared beams for 10 min, and recording both horizontal and vertical activity, under a dim light (Ͻ20 lux). An- 1200/(1200 Ϫ time in the central area). tinociceptive measurements for each mouse were taken 30 min after morning injection by using the tail immersion assay as described previ- The total amount of time spent in the central area was similar in all ously (Janssen et al., 1963). Antinociceptive responses were also evalu- groups of the different experiments (see Statistics). A place conditioning ated in the hot plate test (Columbus Instruments, Columbus, OH) on the score was calculated for each animal as the difference between time spent first day. For the tail immersion, the time to withdraw the tail from the in the drug-paired compartment during the test and preconditioning bath was registered (50 Ϯ 0.5°C), with a cutoff latency of 15 sec to prevent phases. tissue damage. For the hot plate (52 Ϯ 0.5°C), two different nociceptive Statistics. Raw time score values were used for statistical analysis in all thresholds were measured: paw licking (cutoff latency of 30 sec) and the experiments. These values were compared by using two-way ANOVA jumping (cutoff latency of 240 sec). Rectal temperature was measured in between subjects (genotype and treatment) followed by one-way each mouse using an electronic thermocouple flexible rectal probe (Pan- ANOVA for individual differences. Time spent in the drug-paired com- lab, Madrid, Spain). The probe was placed 3 cm into the of the partment during preconditioning in the different groups was compared by mice for 20 sec before the temperature was recorded, and measures were a one-way ANOVA (experimental group as between subjects factor) to taken 40 min after morning injection. ensure use of an unbiased procedure (experiment with MOR Ϫ/Ϫ mice: ϭ ϭ Ϫ Ϫ ϭ On the sixth day, 4 hr after the last THC or vehicle injection, mice were F(5,75) 0.414, p 0.837; experiment with DOR / mice: F(5,64) ϭ Ϫ Ϫ ϭ ϭ placed in a circular clear plastic observation area (30 cm diameter, 80 cm 0.753, p 0.587; experiment with KOR / mice: F(5,60) 0.420, p Ϫ Ϫ ϭ height) for a 15 min period of observation. Body weight and rectal 0.833; experiment with KOR / mice without priming: F(3,37) 0.05, temperature were consecutively measured, and animals received admin- p ϭ 0.985). To verify any possible influence of the mutation in mouse istration of SR141716A (10 mg/kg, i.p.). Mice were then replaced in the spontaneous behavior in the place conditioning paradigm, time spent observation area and observed for 45 min. Measurement of somatic signs in the central compartment during preconditioning in the different before and after SR 141716A challenge were divided in 5 min time groups was also compared by two-way ANOVA between subjects intervals, as previously described (Hutcheson et al., 1998). The number (genotype and treatment). No significant effect of genotype (experi- Ϫ Ϫ ϭ ϭ of bouts of sniffing, writhing, wet dog shakes, and front paw were ment with MOR / mice: F(1,81) 0.017, p 0.897; experiment with Ϫ Ϫ ϭ ϭ Ϫ Ϫ counted. Penile licking or erection, ataxia, hunched posture, tremor, DOR / mice: F(1,70) 1.136, p 0.290; experiment with KOR / ϭ ϭ , and piloerection were scored 1 for appearance and 0 for nonap- mice: F(1,68) 1.377, p 0.260), treatment (experiment with MOR Ϫ Ϫ ϭ ϭ Ϫ Ϫ pearance within each 5 min time period. Scores for the level of activity / mice: F(2,81) 1.757, p 0.180; experiment with DOR / mice: ϭ ϭ ϭ Ϫ Ϫ ϭ were made by giving in each 5 min period a value of 0 low activity F(2,70) 0.372, p 0.372; experiment with KOR / mice: F(2,68) (less than five complete crossings of the observation area), 1 ϭ normal 3.905, p ϭ 0.053) nor interaction between these two factors (experi- Ϫ Ϫ ϭ ϭ activity (between five and twenty complete crossings of the observation ment with MOR / mice: F(2,81) 0.504, p 0.606; experiment with ϭ Ϫ Ϫ ϭ ϭ Ϫ Ϫ area), or 2 increased activity (more than twenty complete crossings of DOR / mice: F(2,70) 0.657, p 0.522; experiment with KOR / ϭ ϭ the observation area). A quantitative value was calculated in each animal mice: F(2,68) 2.060, p 0.136) was observed in any experiment. for the different checked signs by adding the scores obtained in each 5 Individual comparisons of time spent in the drug-paired compartment min time period. A global withdrawal score, ranging from 0 to 100, was during preconditioning and test phases were made with paired two- calculated for each animal by giving to each individual sign a relative tailed Student’s t test. 1148 J. Neurosci., February 1, 2002, 22(3):1146–1154 Ghozland et al. • Cannabinoid Responses in Opioid Knock-Out Mice

Table 1. Three-way ANOVA of hypothermia, hypolocomotion, and antinociception induced during chronic THC treatment in mice lacking ␮-, ␦-, or ␬-opioid receptor

MOR DOR KOR FpFpFp Temperature ϭ Ͻ ϭ Ͻ ϭ Ͻ Day (D) F(5,45) 31.32 p 0.001 F(5,33) 25.09 p 0.001 F(5,36) 53.27 p 0.001 ϭ Ͻ ϭ Ͻ ϭ Ͻ Treatment (T) F(1,49) 152.8 p 0.001 F(1,37) 97.07 p 0.001 F(1,40) 172.3 p 0.001 ϭ ϭ ϭ ϭ ϭ ϭ Genotype (G) F(1,49) 0.685 p 0.412 F(1,37) 0.017 p 0.895 F(1,40) 1.132 p 0.294 ϫ ϭ Ͻ ϭ Ͻ ϭ Ͻ D T F(5,45) 25.92 p 0.001 F(5,33) 22.72 p 0.001 F(5,36) 57.60 p 0.001 ϫ ϭ ϭ ϭ ϭ ϭ ϭ D G F(5,45) 1.713 p 0.151 F(5,33) 0.631 p 0.677 F(5,36) 1.142 p 0.356 ϫ ϭ ϭ ϭ ϭ ϭ ϭ T G F(1,49) 0.434 p 0.513 F(1,37) 0.124 p 0.727 F(1,40) 1.134 p 0.293 ϫ ϫ ϭ ϭ ϭ ϭ ϭ ϭ D T G F(5,45) 0.881 p 0.502 F(5,33) 0.547 p 0.739 F(5,36) 1.265 p 0.300 Locomotor activity ϭ Ͻ ϭ Ͻ ϭ Ͻ Day (D) F(5,32) 13.82 p 0.001 F(5,22) 7.223 p 0.001 F(5,34) 25.11 p 0.001 ϭ Ͻ ϭ Ͻ ϭ Ͻ Treatment (T) F(1,36) 16.84 p 0.001 F(1,26) 5.325 p 0.05 F(1,38) 20.26 p 0.001 ϭ Ͻ ϭ ϭ ϭ ϭ Genotype (G) F(1,36) 7.216 p 0.05 F(1,26) 3.287 p 0.081 F(1,38) 0.185 p 0.669 ϫ ϭ Ͻ ϭ Ͻ ϭ Ͻ D T F(5,32) 6.845 p 0.001 F(5,22) 4.833 p 0.01 F(5,34) 9.164 p 0.001 ϫ ϭ ϭ ϭ ϭ ϭ Ͻ D G F(5,32) 2.014 p 0.103 F(5,22) 0.851 p 0.529 F(5,34) 1.288 p 0.05 ϫ ϭ ϭ ϭ ϭ ϭ ϭ T G F(1,36) 3.473 p 0.071 F(1,26) 0.072 p 0.727 F(1,38) 0.700 p 0.408 ϫ ϫ ϭ ϭ ϭ ϭ ϭ ϭ D T G F(5,32) 0.509 p 0.768 F(5,22) 0.905 p 0.495 F(5,34) 0.830 p 0.882 Tail immersion ϭ Ͻ ϭ Ͻ ϭ Ͻ Day (D) F(5,43) 9.615 p 0.001 F(5,35) 3.987 p 0.01 F(5,37) 12.15 p 0.001 ϭ Ͻ ϭ Ͻ ϭ Ͻ Treatment (T) F(1,47) 71.10 p 0.001 F(1,39) 65.76 p 0.001 F(1,41) 85.28 p 0.001 ϭ ϭ ϭ ϭ ϭ ϭ Genotype (G) F(1,47) 0.372 p 545 F(1,39) 0.178 p 0.675 F(1,41) 3.397 p 0.073 ϫ ϭ Ͻ ϭ Ͻ ϭ Ͻ D T F(5,43) 14.99 p 0.01 F(5,35) 6.987 p 0.001 F(5,37) 13.428 p 0.001 ϫ ϭ ϭ ϭ ϭ ϭ ϭ D G F(5,43) 1.150 p 0.349 F(5,35) 1.823 p 0.134 F(5,37) 1.587 p 0.188 ϫ ϭ ϭ ϭ ϭ ϭ ϭ T G F(1,47) 0.261 p 0.612 F(1,39) 1.081 p 0.305 F(1,41) 1.825 p 0.184 ϫ ϫ ϭ ϭ ϭ ϭ ϭ ϭ D T G F(5,43) 1.195 p 0.328 F(5,35) 0.823 p 0.542 F(5,37) 0.659 p 0.657

Three-way ANOVA repeated measures with treatment and genotype as between-subjects factors and day as within-subjects factor. See Materials and Methods for details. Overall significant effects of day and treatment and interactions between these two factors are observed in the three strains of mice. Main significant effect of genotype is only observed for locomotor activity in the MOR strain, and significant interaction between day and genotype is only observed for locomotor activity of the KOR strain.

RESULTS developed similarly in all mouse genotypes. Tolerance to an- THC tolerance and withdrawal in MOR ؊/؊, DOR ؊/؊, tinociceptive effects of THC was also similar in all genotypes .(and KOR ؊/؊ mice (Table 1, Fig. 1 To induce tolerance and dependence, we chronically injected a We then administered the cannabinoid antagonist SR141716A high dose of THC (20 mg/kg). The first THC injection produced (10 mg/kg) in chronically THC-treated mice. A significant ex- similar antinociception in wild-type, MOR Ϫ/Ϫ, DOR Ϫ/Ϫ, and pression of several signs of withdrawal, including wet dog shakes, KOR Ϫ/Ϫ mice, in both the hot plate (paw licking: mean value, paw tremor, ptosis, piloerection, mastication, and sniffing, was Ϯ ϭ ϭ detected in wild-type, MOR Ϫ/Ϫ, DOR Ϫ/Ϫ, and KOR Ϫ/Ϫ 79.3 3.26% of analgesia, F(5,68) 0.788, p 0.562; jump: mean Ϯ ϭ ϭ mice. Expression of these signs was comparable in mutant ani- value, 95.9 1.22% of analgesia, F(5,68) 0.913, p 0.478) and the tail immersion (mean value, 47.8 Ϯ 5.14% of analgesia; mals and their respective wild-type controls, except for paw Ϫ Ϫ F ϭ 1.386; p ϭ 0.24). This first THC injection also produced tremor that was significantly reduced in THC-treated DOR / (5,68) ϩ ϩϭ Ϯ hypolocomotion (mean decrease, 66.2 Ϯ 2.04%; F ϭ 0.867; mice (vehicle-treated DOR / 2.46 1.39; vehicle-treated (5,67) Ϫ Ϫϭ Ϯ ϩ ϩϭ Ϯ p ϭ 0.508) and hypothermia (mean decrease, 3.63 Ϯ 0.34°C; DOR / 3.25 1.13; THC-treated DOR / 81.6 9.78; Ϫ Ϫϭ Ϯ F ϭ 2.091; p ϭ 0.077) that did not differ between genotypes. THC-treated DOR / 64.4 13.4; two-way ANOVA, ge- (5,67) ϭ Ͻ ϭ Ͻ During repeated THC administration, a progressive decrease notype: F(1,38) 76.97, p 0.001; treatment: F(1,38) 5.173, p ϫ ϭ Ͻ in the antinociceptive, hypolocomotor, and hypothermic activity 0.05, genotype treatment: F(1,38) 5.556, p 0.05; one-way ϭ Ͻ of the drug was observed. This tolerance developed similarly in ANOVA for genotype comparison: F(1,14) 4.894, p 0.05). wild-type, MOR Ϫ/Ϫ, and DOR Ϫ/Ϫ mice, whereas minor Other signs of THC withdrawal such as body tremor, writhing, changes were observed in KOR Ϫ/Ϫ mice (Table 1, Fig. 1). hunched posture, and penile licking were less obvious, and no Tolerance for locomotor responses was developed on the second difference was detectable in any of these signs when comparing day of THC treatment in all groups. However, habituation in mutant and wild-type groups. We determined global withdrawal vehicle-treated mice during the first3doftesting was observed. scores for each MOR Ϫ/Ϫ, DOR Ϫ/Ϫ, and KOR Ϫ/Ϫ genotype This habituation can also be important for reducing initial differ- and their wild-type controls. Total withdrawal score comparisons ences between THC and vehicle-treated groups. Noticeable was revealed no significant alteration of THC withdrawal in any group the significant lower locomotion in THC-treated KOR Ϫ/Ϫ of mutant mice (Fig. 2). Indeed, two-way ANOVA revealed a mice compared with THC-treated wild-type controls on day 5 significant effect of THC treatment (experiment with MOR Ϫ/Ϫ ϭ Ͻ ϭ Ͻ Ϫ Ϫ (F(1,21) 13.426; p 0.005). Tolerance to the hypothermic effects mice: F(1,41) 123.2, p 0.001; experiment with DOR / mice: Ghozland et al. • Cannabinoid Responses in Opioid Knock-Out Mice J. Neurosci., February 1, 2002, 22(3):1146–1154 1149

Ϫ/Ϫ and KOR Ϫ/Ϫ mutants receiving the previous THC single injection (Tables 2–4, Fig. 3). In contrast, this response was completely absent in MOR Ϫ/Ϫ mice (Tables 2–4, Fig. 3), dem- onstrating that ␮-opioid receptors are essential for the rewarding effects of THC. We then used another place conditioning protocol where ani- mals received no injection of THC before conditioning and were subjected to a high dose of THC (5 mg/kg). Under those condi- tions only THC dysphoria is detectable. In agreement with pre- vious studies (Valjent and Maldonado, 2000), wild-type mice showed marked conditioned place aversion, as revealed by signif- icant decrease in time spent in the drug-paired compartment in MOR ϩ/ϩ, DOR ϩ/ϩ, and KOR ϩ/ϩ (Table 4), and in the score values (Tables 2, 3, Fig. 3). In DOR Ϫ/Ϫ mice, conditioned place aversion to THC (5 mg/kg) was comparable with wild-type (Tables 2–4, Fig. 3). In MOR Ϫ/Ϫ mutants, the aversive effects of THC were diminished but not abolished (Tables 2–4, Fig. 3). In KOR Ϫ/Ϫ animals, THC place aversion was completely absent (Tables 2–4, Fig. 3). The latter finding demonstrates that ␬ receptors are critically implicated in the dysphoric aspect of THC activity. Because this is a first evidence for an involvement of ␬ recep- tors in THC aversive properties, we further explored this finding using a third place conditioning protocol. Mice were conditioned to 1 mg/kg of THC without receiving any injection of the drug Figure 1. Adaptive responses to chronic THC in KOR Ϫ/Ϫ mice. De- before the conditioning period. We have previously shown that velopment of tolerance to the antinociceptive, hypothermic, and hypolo- this protocol does not reveal any motivational effect of THC in comotor effects of chronic THC (20 mg/kg, i.p., twice daily) is minimally wild-type mice, most probably because THC reward and dyspho- affected in KOR Ϫ/Ϫ mice. Number of mice per group from 10 to 14. Values are expressed as means Ϯ SEM. ૺp Ͻ 0.05; ૺૺp Ͻ 0.01; ૺૺૺp Ͻ ria equally oppose each other (Valjent and Maldonado, 2000). 0.001; comparison between treatments (one-way ANOVA); ૾૾p Ͻ 0.01, Accordingly, wild-type mice showed no place conditioning, as comparison between genotypes (one-way ANOVA).

ϭ Ͻ Ϫ Ϫ F(1,38) 241.3, p 0.001; experiment with KOR / mice: F(1,36) ϭ 44.31, p Ͻ 0.001), but not effect of genotype (experiment with Ϫ Ϫ ϭ ϭ MOR / mice: F(1,41) 0.122, p 0.873; experiment with DOR Ϫ Ϫ ϭ ϭ Ϫ Ϫ / mice: F(1,38) 0.135, p 0.715; experiment with KOR / ϭ ϭ mice: F(1,36) 0.092, p 0.736) nor interaction between treatment Ϫ Ϫ ϭ and genotype (experiment with MOR / mice: F(1,41) 0.058, ϭ Ϫ Ϫ ϭ ϭ p 0.810; experiment with DOR / mice: F(1,38) 0.525, p Ϫ Ϫ ϭ ϭ 0.525; experiment with KOR / mice: F(1,36) 0.019, p 0.891) in the three experiments. THC-induced place conditioning We explored motivational responses to THC in MOR Ϫ/Ϫ, DOR Ϫ/Ϫ, and KOR Ϫ/Ϫ mice, using several place conditioning pro- tocols. We first investigated the rewarding properties of THC, in a place conditioning protocol that minimizes the aversive activity of THC. Mice received a single injection of a low dose of THC (1 mg/kg) in their home cage and were subsequently conditioned to the same dose of the drug. Time spent by all the mice in the two conditioning compartments of the apparatus was similar during the preconditioning phase in the different experiments (mean in striped compartment: 615.8 sec; mean in dotted compartment: 582.6 sec). No initial place preference or aversion was observed in any of the experiments. Under those conditions, all the groups of wild-type mice showed a strong place preference, as previously reported (Valjent and Maldonado, 2000). This was revealed by Figure 2. Somatic expression of withdrawal from THC after SR 141716A significant increases in the time spent in the drug-paired com- administration (10 mg/kg, i.p.) is similar in MOR Ϫ/Ϫ, DOR Ϫ/Ϫ, and partment from the preconditioning to the test phase in MOR KOR Ϫ/Ϫ mice and in their respective wild-type controls. Mice received ϩ/ϩ, DOR ϩ/ϩ, and KOR ϩ/ϩ mice (see Table 4), as well as by a chronic administration of vehicle or THC (20 mg/kg, i.p., twice daily) for 6 d. Values are expressed as mean Ϯ SEM of global withdrawal scores significant scores when comparing THC-treated mice with the calculated by giving to each individual sign a proportional weight. Num- respective vehicle controls (Tables 2, 3, Fig. 3). A similar condi- ber of mice per group from 10 to 14. ૺૺૺp Ͻ 0.001, comparison between tioned place preference to THC (1 mg/kg) was observed in DOR treatments (one-way ANOVA). 1150 J. Neurosci., February 1, 2002, 22(3):1146–1154 Ghozland et al. • Cannabinoid Responses in Opioid Knock-Out Mice

Table 2. Two-way ANOVA of place conditioning scores to THC of mice lacking ␮-, ␦-, or ␬-opioid receptor

MOR DOR KOR FpFpFp ϭ ϭ ϭ ϭ ϭ ϭ Genotype (G) F(1,81) 0.503 p 0.480 F(1,70) 0.02 p 0.887 F(1,68) 2.879 p 0.095 ϭ Ͻ ϭ Ͻ ϭ Ͻ Treatment (T) F(1,81) 10.04 p 0.001 F(2,70) 30.66 p 0.001 F(2,68) 21.261 p 0.001 ϫ ϭ Ͻ ϭ ϭ ϭ Ͻ G T F(2,81) 6.516 p 0.01 F(2,70) 0.351 p 0.266 F(2,68) 3.391 p 0.05

Two-way ANOVA with treatment and genotype as between-subjects factors. See Materials and Methods for details. Overall significant effects of treatment are observed in the three strains of mice. A significant interaction between genotype and treatment is observed in MOR and KOR strains.

Table 3. One-way ANOVA of place conditioning scores to THC of mice lacking ␮-, ␦-, or ␬-opioid receptor

MOR DOR KOR Group Comparison FpFpFp ϩ ϩ Ϫ Ϫ ϭ ϭ ϭ ϭ ϭ ϭ Vehicle / vs / F(1,27) 0.003 p 0.955 F(1,18) 0.473 p 0.501 F(1,19) 0.017 p 0.898 ϩ ϩ Ϫ Ϫ ϭ Ͻ ϭ ϭ ϭ ϭ THC1 / vs / F(1,26) 12.76 p 0.001 F(1,24) 2.705 p 0.114 F(1,22) 0.046 p 0.833 ϩ ϩ Ϫ Ϫ ϭ ϭ ϭ ϭ ϭ Ͻ THC5 / vs / F(1,25) 2.292 p 0.143 F(1,25) 0.131 p 0.721 F(1,21) 9.995 p 0.01 ϩ ϩ ϭ Ͻ ϭ Ͻ ϭ Ͻ / THC1 vs VEHICLE F(1,26) 8.953 p 0.01 F(1,21) 4.088 p 0.05 F(1,22) 8.041 p 0.05 ϩ ϩ ϭ Ͻ ϭ Ͻ ϭ Ͻ / THC5 vs VEHICLE F(1,26) 5.752 p 0.05 F(1,21) 7.785 p 0.05 F(1,23) 9.704 p 0.01 Ϫ Ϫ ϭ ϭ ϭ Ͻ ϭ ϭ / THC1 vs VEHICLE F(1,27) 0.124 p 0.727 F(1,21) 14.437 p 0.001 F(1,21) 7.165 p 0.05 Ϫ Ϫ ϭ ϭ ϭ ϭ ϭ ϭ / THC5 vs VEHICLE F(1,26) 0.914 p 0.348 F(1,22) 1.952 p 0.177 F(1,20) 0.13 p 0.910

One-way ANOVA with treatment or genotype as between-subjects factors. See Materials and Methods for details. Significant effects of genotype are observed in the MOR strain at 1 mg/kg THC and in the KOR strain at 5 mg/kg THC. The significant effect of treatment observed in MOR ϩ/ϩ mice is absent in MOR Ϫ/Ϫ mice with 1 mg/kg THC. The significant effect of treatment observed in KOR ϩ/ϩ mice is absent in KOR Ϫ/Ϫ mice with 5 mg/kg THC.

Figure 3. THC place preference and aversion. A, Place preference to THC is abolished in MOR Ϫ/Ϫ mice, whereas place aversion is diminished (number of mice per group from 13 to 14); B, DOR Ϫ/Ϫ mice show similar place conditioning to THC than their wild-type controls (number of mice per group from 10 to 13); and C, Place aversion to THC is abolished in KOR Ϫ/Ϫ mice (number of mice per group from 11 to 12) mice. THC1 (1 mg/kg, i.p.); THC5 (5 mg/kg, i.p.). Mice conditioned to the dose of 1 mg/kg THC received a previous THC injection (1 mg/kg, i.p.) in their home cage, 24 hr before the start of the conditioning phase. In wild-type mice, THC1 conditions reveal THC place preference, whereas THC5 conditions show THC place aversion. Values are expressed as mean Ϯ SEM. Scores were calculated as the difference between test and preconditioning time spent in the drug-paired compartment. ૺp Ͻ 0.05, ૺૺp Ͻ 0.01, comparison between treatments; ૾૾p Ͻ 0.01, comparison between genotypes (one-way ANOVAs). shown by comparable time spent in the drug-paired compartment those conditions, ␬ receptor activity hinders the rewarding prop- ϭϪ ϭ between preconditioning and test (t(1,9) 0.680; p 0.514) and erties of THC in wild-type mice. comparable score values between animals conditioned to THC or ϭ ϭ Ϫ Ϫ vehicle (F(1,19) 0.348; p 0.562) (Fig. 4). In contrast, KOR / DISCUSSION mice conditioned to 1 mg/kg of THC showed under these exper- Bidirectional interactions between the opioid and cannabinoid imental conditions a significant place preference as seen by an systems have been reported (Manzanares et al., 1999). Here we ϭ ␮ ␦ ␬ increase in the time spent in the drug-paired compartment (t(1,9) show that disruption of -, -, or -opioid receptor gene does not Ϫ2.770; p Ͻ 0.05) and a significantly different conditioning score modify acute THC responses or the expression of THC with- ϭ Ͻ from vehicle controls (F(1,18) 5.587; p 0.05). Also, although drawal, and that the development of THC tolerance is only Ϫ Ϫ ␮ ␬ vehicle groups from both genotypes showed similar scores (F(1,18) slightly altered in KOR / mice. Both - and -opioid ligands ϭ 0.006; p ϭ 0.938), KOR Ϫ/Ϫ animals conditioned to THC have been previously reported to modulate cannabinoid antino- displayed significantly different scores from wild-type controls ciception (Manzanares et al., 1999). Thus, THC antinociception ϭ Ͻ ␬ ␬ (F(1,19) 4.877; p 0.05). Therefore, the lack of receptors in was blocked in mice by the -selective opioid antagonist norbin- mutant mice reveals THC place preference, suggesting that, under altorphimine and by high doses of the nonselective opioid antag- Ghozland et al. • Cannabinoid Responses in Opioid Knock-Out Mice J. Neurosci., February 1, 2002, 22(3):1146–1154 1151

Table 4. Paired Student’s t test comparisons of the time spent in the drug-paired compartment during the preconditioning and test phases, in mice lacking ␮-, ␦-, or ␬-opioid receptor

MOR pre-cond test tp ϩ ϩ Ϯ Ϯ ϭ ϭ / VEHICLE 677 63 672 92 t(1,13) 0.075 p 0.942 Ϯ Ϯ ϭϪ Ͻ THC1 589 64 874 71 t(1,12) 6.958 p 0.01 Ϯ Ϯ ϭ Ͻ THC5 651 58 296 76 t(1,12) 2.932 p 0.05 Ϫ Ϫ Ϯ Ϯ ϭ ϭ / VEHICLE 607 65 611 79 t(1,13) 0.076 p 0.941 Ϯ Ϯ ϭ ϭ THC1 615 65 573 71 t(1,13) 0.477 p 0.641 Ϯ Ϯ ϭ ϭ THC5 574 51 394 77 t(1,12) 1.091 p 0.297

DOR pre-cond test tp ϩ ϩ Ϯ Ϯ ϭ ϭ / VEHICLE 734 66 733 75 t(1,8) 0.035 p 0.973 Ϯ Ϯ ϭϪ Ͻ THC1 590 62 780 63 t(1,12) 3.739 p 0.01 Ϯ Ϯ ϭ Ͻ THC5 589 45 359 71 t(1,12) 3.253 p 0.01 Ϫ Ϫ Ϯ Ϯ ϭ ϭ / VEHICLE 582 91 576 107 t(1,9) 0.070 p 0.945 Ϯ Ϯ ϭϪ Ͻ THC1 578 61 879 53 t(1,11) 4.443 p 0.001 Ϯ Ϯ ϭ Ͻ THC5 620 54 330 79 t(1,12) 3.31 p 0.01

KOR pre-cond test tp ϩ ϩ Ϯ Ϯ ϭ ϭ / VEHICLE 612 57 606 51 t(1,11) 1.013 p 0.333 Ϯ Ϯ ϭϪ Ͻ THC1 531 87 745 86 t(1,10) 2.971 p 0.05 Ϯ Ϯ ϭ Ͻ THC5 663 69 308 72 t(1,11) 4.142 p 0.01 Ϫ Ϫ Ϯ Ϯ ϭ ϭ / VEHICLE 604 106 572 89 t(1,9) 0.022 p 0.983 Ϯ Ϯ ϭϪ Ͻ THC1 573 66 788 90 t(1,11) 3.670 p 0.01 Ϯ Ϯ ϭ ϭ THC5 563 45 493 67 t(1,10) 0.343 p 0.739

Values of time spent in the drug-paired compartment during the preconditioning and test phases are expressed in seconds. Paired Student’s t test was used to compare the time spent in the drug-paired side during preconditioning and test phases within each group of mice. See Materials and Methods for details. The significant differences between time spent in the drug-paired compartment during the preconditioning and test phases observed in MOR ϩ/ϩ mice with 1 and 5 mg/kg THC were abolished in MOR Ϫ/Ϫ mice. The significant difference between time spent in the drug-paired compartment during the preconditioning and test phases observed in KOR ϩ/ϩ mice with 5 mg/kg THC was abolished in KOR Ϫ/Ϫ mice. onist naloxone (Welch, 1993; Smith et al., 1998). The synergistic displayed tolerance to the effects of ␬ selective agonists (U-50,488 effects of morphine and THC on antinociception were also and CI-977), but not of ␮ or ␦ selective agonists (Smith et al., 1994). blocked by both and ␤-funaltrexamine, a ␮ The lack of alteration of THC withdrawal was unexpected, selective opioid antagonist (Reche et al., 1996). The absence of because clear interactions between opioid and cannabinoid de- changes in THC antinociception in this study is unlikely attrib- pendence have been reported. Thus, naloxone, the prototypic utable to a ceiling effect, because a maximal response was ob- nonselective opioid antagonist, precipitates an opioid-like with- served in one response (jump in the hot plate) over three noci- drawal syndrome in cannabinoid-dependent rodents (Kaymak- ceptive thresholds evaluated. Rather, we may suggest that the calan et al., 1977; Navarro et al., 1998) and conversely, the CB1 ablation of one opioid receptor only is not sufficient to reveal cannabinoid receptor antagonist SR 141716A induces withdrawal significant changes, and indeed, high doses of opioid antagonists in morphine-dependent rats (Navarro et al., 1998). This suggests are usually required to block THC antinociception (Manzanares that simultaneous activation of the two endogenous systems could et al., 1999). THC antinociception in the tail immersion test was participate to both opioid and cannabinoid dependence. Consis- reduced in knock-out mice lacking the preproenkephalin gene tent with this notion, gene-targeting experiments have shown (Valverde et al., 2000), but derivatives from are attenuated naloxone-precipitated opioid withdrawal in morphine- not selective agonists of any opioid receptor. dependent CB1 knock-out mice (Ledent et al., 1999) and atten- Besides, the deletion of ␮-or␦-opioid receptors has no mea- uated SR141716A-precipitated cannabinoid withdrawal in THC- surable consequence on the development of THC tolerance, dependent mice lacking preproenkephalin gene (Valverde et al., whereas tolerance was reduced in KOR Ϫ/Ϫ mice on the last day 2000). Pretreatment with THC (Hine et al., 1975; Valverde et al., of treatment. Although these modifications were subtle, the data 2001) or anandamide (Vela et al., 1995), have been also shown to suggest that ␬ receptors could contribute to the development of decrease morphine withdrawal. Our finding that the disruption of adaptive responses to chronic THC, in agreement with the dem- a single opioid receptor gene has no major consequences on the onstration of cross-tolerance between THC and ␬-opioid agonists somatic expression of THC withdrawal may indicate that concom- (Smith et al., 1994). Besides, THC-induced antinociceptive toler- itant changes in the activity of several opioid receptors are impli- ance can be modified by antisense oligodeoxynucleotides to the ␬ cated in the expression of THC withdrawal. Possibly the analysis of receptor (Rowen et al., 1998), and mice tolerant to THC also combinatorial double or triple opioid receptor-deleted mice may 1152 J. Neurosci., February 1, 2002, 22(3):1146–1154 Ghozland et al. • Cannabinoid Responses in Opioid Knock-Out Mice

tors reveals THC place preference in mutant mice, suggesting that, under those conditions, ␬ receptor activity hinders the rewarding properties of THC in wild-type mice. These observa- tions indicate that an endogenous ␬ receptor tone underlies THC aversion, a hypothesis that has not been explored by pharmaco- logical studies. Several peripheral and somatic effects of THC can participate in its aversive properties, as it has been previously reported with ␬ agonists (Bechara et al., 1987). Indeed, THC produces hypothermia and hypolocomotion that can induce a discomfort state in the mouse, leading to the aversive response. The exact involvement of these somatic responses remains to be clarified. A possible limitation in the interpretation of these results is the required use of ethanol to dissolve THC and SR 141716A. In- deed, ethanol is also a psychoactive compound sharing some common pharmacological properties with cannabinoids and opi- oids. The pharmacological responses induced by these vehicle solutions of cannabinoid agents have been reported to be exclu- sively caused by the activation of CB1 cannabinoid receptors because both acute and chronic THC effects were abolished in Figure 4. KOR Ϫ/Ϫ mice show place preference to THC (1 mg/kg, i.p.), knock-out animals deficient in these CB1 receptors (Ledent et al., without priming exposure, whereas their wild-type controls do not. Val- 1999). However, THC behavioral effects can be modulated by the ues are expressed as mean Ϯ SEM. Number of mice per group from 9 to presence of ethanol, and this modulation may be different in ␮-, 10. Scores calculated as the difference between test and preconditioning ␦ ␬ ૺ Ͻ -, or -opioid receptor knock-out mice. Therefore, a possible time spent in the drug-paired compartment. p 0.05, comparison contribution of ethanol in the behavioral responses observed in between treatments; ૾p Ͻ 0.05, comparison between genotypes (one-way ANOVAs). these knock-out mice cannot be excluded. Ethanol was also present in the SR 141716A solution used to precipitate THC withdrawal. The behavioral signs of cannabinoid withdrawal ob- reveal a concerted implication of several components of the opioid served when using this procedure are caused by the blockade of system in the development of THC physical dependence. CB-1 cannabinoid receptors because they were abolished in CB-1 The suppression of THC rewarding effects in MOR Ϫ/Ϫ mice knockout mice (Ledent et al., 1999), and these behavioral signs provides a clear genetic evidence to clarify the crucial role of ␮ were not elicited by the ethanol-containing vehicle alone (Cook et receptors in THC motivational responses. Gene targeting exper- al., 1998). However, the presence of ethanol could attenuate the iments have recently demonstrated the central role of ␮-opioid behavioral expression of cannabinoid withdrawal, considering the receptors in mediating rewarding properties of several drugs of common pharmacological effects of both compounds. This obser- abuse, including morphine (Matthes et al., 1996), ethanol (Rob- vation must be considered for the interpretation of the cannabi- erts et al., 2000), and now THC (this study). ␮ receptors, there- noid withdrawal results. Besides, the possibility of developmental fore, seem to represent a convergent substrate for positive rein- compensations after the deletion of the different opioid receptors forcement. A possible mechanism to explain the involvement of ␮ and/or influences of the hybrid genetic background (Kelly et al., receptors in cannabinoid rewarding effects could be caused by 1998) cannot be discarded. However, no homologous compensa- their effects on the mesolimbic dopaminergic system. Thus, THC- tory changes have been reported in these knock-out mice. Indeed, induced increase in mesolimbic dopaminergic activity (Navarro the abolition of ␮-opioid (Matthes et al., 1996), ␦-opioid (Filliol et et al., 1993) was reversed by naloxone (Chen et al., 1990), or al., 2000), or ␬-opioid (Simonin et al., 1998) receptors has no ␮ , a 1 antagonist (Tanda et al., 1997). However, influence on the binding properties and distribution of the other another study showed no effect of naloxone on THC excitatory opioid receptors, and did not modify the expression of the differ- activity on neurons from the ent precursors. (French, 1997). Therefore, other mechanisms could also mediate The present novel finding highlights the involvement of homeo- the implication of ␮-opioid receptors in cannabinoid motivational static opioid mechanisms in cannabinoid motivational responses. properties. Interestingly, morphine-induced rewarding effects We propose that opposing ␮-opioid and ␬-opioid receptor activ- were suppressed in mice deficient in CB1 cannabinoid receptors ities mediate the dual euphoric–dysphoric effects of THC. This (Ledent et al., 1999), suggesting a bidirectional influence of hypothesis is consistent with the proposed opposite role of the ␮-opioid and CB1 cannabinoid receptors on reward processes. two opioid receptors in modulating mesolimbic dopaminergic ␦-opioid receptors do not seem to be involved in the motiva- activity (Di Chiara, 1995) and suggests that THC influences both tional responses to THC. The differential location of CB1 canna- players within the reward circuitry. A possible mechanism could binoid and ␦ opioid receptors may be important for this result. be that cannabinoid receptor activation modifies endogenous Although both receptors are localized in the opioid peptide levels in mesolimbic areas that would, in turn, (Mansour et al., 1995; Robbe et al., 2001), they might not be modulate dopaminergic activity. This is supported by findings of colocalized in other brain regions implicated in the rewarding increased opioid levels in the (Corchero effects of cannabinoids. The present results demonstrate a crucial et al., 1997a,b), or preproenkephalin mRNA levels in the striatum involvement of ␬-opioid receptors in the aversive properties of and nucleus accumbens (Manzanares et al., 1998) after cannabi- THC. Thus, THC-induced conditioned place aversion was abol- noid treatment. Cannabinoids and opioids might also interact at ished in KOR Ϫ/Ϫ mice. In addition, the lack of ␬-opioid recep- the level of their signaling activity. 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